A novel Carcinoembryonic Antigen (CEA)-Targeted Trimeric Immunotoxin shows significantly enhanced Antitumor Activity in Human Colorectal Cancer Xenografts

Abstract

Immunotoxins are chimeric molecules, which combine antibody specificity to recognize and bind with high-affinity tumor-associated antigens (TAA) with the potency of the enzymatic activity of a toxin, in order to induce the death of target cells. Current immunotoxins present some limitations for cancer therapy, driving the need to develop new prototypes with optimized properties. Herein we describe the production, purification and characterization of two new immunotoxins based on the gene fusion of the anti-carcinoembryonic antigen (CEA) single-chain variable fragment (scFv) antibody MFE23 to α-sarcin, a potent fungal ribotoxin. One construct corresponds to a conventional monomeric single-chain immunotoxin design (IMTXCEAαS), while the other one takes advantage of the trimerbody technology and exhibits a novel trimeric format (IMTXTRICEAαS) with enhanced properties compared with their monomeric counterparts, including size, functional affinity and biodistribution, which endow them with an improved tumor targeting capacity. Our results show the highly specific cytotoxic activity of both immunotoxins in vitro , which was enhanced in the trimeric format compared to the monomeric version. Moreover, the trimeric immunotoxin also exhibited superior antitumor activity in vivo in mice bearing human colorectal cancer xenografts. Therefore, trimeric immunotoxins represent a further step in the development of next-generation therapeutic immunotoxins.

Novel cucurmosin‐based immunotoxin targeting programmed cell death 1‐ligand 1 with high potency against human tumor in vitro and in vivo

“…Although D‐CUS 245C showed a clear inhibition of tumor growth in s.c. tumor‐bearing xenograft models, poor penetration for D‐CUS 245C into the tumor local region is the main obstacle to the treatment of solid tumors. The emergence of recombinant technologies has revolutionized the production of mAbs 39 . Currently, most of the ITs in clinical trials have been designed as recombinant fusion proteins linking to different Ab fragments, such as a flexible peptide, a disulfide bridge, or both, with reduced size and improved tumor penetration, to achieve greater specificity and antitumor efficacy 4,40,41 .…”

Inclusion of a Furin Cleavage Site Enhances Antitumor Efficacy against Colorectal Cancer Cells of Ribotoxin α-Sarcin- or RNase T1-Based Immunotoxins

“…This work not only represents a step forward in optimizing the cytotoxic efficacy of immunotoxins based on α-sarcin and RNase T1, but also highlights the development of an immunotoxin design platform based on these ribonucleases, including new designs with different specificities, with monomeric or trimeric formats [74,75], or including the use of non-immunogenic variants of α-sarcin [38]. The combination of all these optimization approaches will represent a very important boost for its application in the clinic.…”

Colorectal Cancer Immunotherapy: Options and Strategies

“…This leads to highly targeted antigen recognition and allows active trafficking to tumor sites, in vivo expansion and long-term persistence. Usually, CAR-T cells are engineered toward tumor-associated antigens (TAAs) such as CD19 in diffuse large B cell lymphoma (DLBCL) ( 129 ), interleukin 13 receptor alpha-2 (IL13Rα2) ( 130 ) and epidermal growth factor variant III (EGFRvIII) in glioblastoma ( 131 ) and carcinoembryonic antigen (CEA) in colorectal cancer ( 132 ) to promote cytotoxicity and apoptosis ( Figure 3 ). The advantage of CAR-T cells is their specificity in targeting cell surface TAA in an MHC-independent manner.…”